The use of dot assays and Western blots have become widespread in biochemical laboratories. In many cases, their use has been limited to screening methods for immunoreactive proteins. There is an increasing trend towards quantitation using these methods by autoradiography or dye-based densitometry. However, the problem of determining the total protein content on nitrocellulose matrices has only recently received any attention which is due, in part, to the development of new protein assay methods. Such determinations have considerable value for a variety of applications.
In recent years, a number of new methods for the assay of protein have been published that offer either increased sensitivity or facility over older methods like the Biuret method (Itzhaki, R.F., et al., Anal. Biochem. 9:401-410 (1964)) and Lowry's method (Lowry, O.H., et al., J. Biol. Chem. 193:265-275 (1951)). Notably, the method introduced by Bradford (Anal. Biochem. 72:248-254 (1976)) has been popular. This method exploits the protein-binding properties of dyes, which have long been utilized in gel electrophoresis, for the visualization of protein bands and, more recently, in differential adsorption of proteins by dye adsorption chromatography (see Scopes, R.K., Anal. Biochem. 136:525-529 (1984) for example). Variations of the Bradford assay are reported for the quantitation of proteins immobilized on immunoadsorbents by elution of protein-bound dye under harsh conditions. Ahmad, H., et al., Anal. Biochem. 48:533-541 (1985).
Krystal et al., Anal. Biochem. 48:451-460 (1985), report a sensitive protein stain for the assay of protein solutions. This assay permits nanogram quantities of proteins to be determined. High sensitivity is critical for the quantitation of proteins, produced from large-scale purifications, which may be present in the order of micrograms. However, the Krystal assay suffers from being prone to rather a large number of interfering substances common to biochemical samples, which require removal before the assay.
Interest in the use of protein binding dyes for the assay and affinity purification of proteins has spurred new methodologies. Kaplan and Pedersen, Anal. Biochem. 150:97-104 (1985) report a modification of an earlier procedure (Schaffner, W., et al., Anal. Biochem. 56:502-514 (973)) using amido black dye. Another dye-binding protein assay was developed by Redinbaugh and Campbell (Anal. Biochem. 147:144-147 (1985)) which utilized microtiter plates in a similar manner to ELISAs.
Since polyvinyl chloride (microtiter plates) became predominantly replaced by nitrocellulose (and other matrices like nylon and diazotized cellulose) for protein adsorption (Gershoni, J.M., et al., Anal. Biochem. 131:1-15 (1983)), the use of immunodot assays on nitrocellulose has become widespread (Hawkes, R., et al., Anal. Biochem. 119:142-147 (1982) and Suresh, M.R., et al., Anal. Biochem. 151:192-195 (1985)). Nakarmur et al. (Anal. Biochem. 311-319 (1985)) reported a similar method of spotting nitrocellulose with protein, and staining the total protein thus adsorbed with dye (amido black or Ponceau red), with subsequent densitometry. This method was extremely rapid, sensitive, and, unlike spectrophotometric proteins assays, was almost without interference from common laboratory chemicals. This method allows samples containing 0.05-10 ug to be detected (a 200 fold range). Another such assay was disclosed by Sportsman, J.R., et al., Anal. Biochem. 139:298-300 (1984), which involved detection by laser densitometry. However, different sample proteins give standard curves with different slopes. These assays are affected by both differential adhesion of proteins to nitrocellulose and differential dye-binding properties of each sample protein. Thus, differential protein adhesion remains a limitation of such nitrocellulose-based protein assays.
Protein assays using nitrocellulose filters have also been reported by Kuno, H., et al., Nature (London) 215:974-975 (1967); Bramhall, S. et al., Anal. Biochem. 31:146-148 (1969); and Pristoupil, T.I. et. al., Nature (London) 212:75-76 (1966). These assays involve elution of stained protein samples for spectrophotometry (Kuno, supra), or calculation of the area of each stained spot (Pristoupil, suora). Both of these quantitation methods limit the usefulness of the method.
Kumar, B.V., et al., Biochem. Biophys. Res. Commun. 131:883-891 (1985) disclose quantitation of proteins on nitrocellulose by iodination with chloramine-T and potassium iodide. The bound iodine is then detected with starch solution. Hancock, K., et al., Anal. Biochem. 33:157-162 (1983), disclose a method for the qualitative determination of proteins on Western blots by transfer onto nitrocellulose and staining with india ink. Yugn, K.C., et al., Anal. Biochem. 126:398-402 (1982), disclose a method for the detection of nanogram quantities of proteins by transfer of the protein from a Western blot to nitrocellulose, followed by staining with a silver solution. Kittler, J.M., et al., Anal. Biochem. 137:210-216 (1984), disclose an immunochemical method for detecting proteins on blots which involves derivatizing the protein with a group recognized by an antibody, followed by detection. However, all of these methods suffer from the disadvantage that the sample protein is chemically modified by the assay, thus making recovery of the sample protein either very difficult or impossible.
Another method for quantitation of proteins on nitrocellose is taught by Wolff, et al., Anal. Biochem. 147:396-400 (1985). In this method, TNP (2,4,6-trinitrobenzene sulfonic acid) is used to derivatize the proteins that are immobilized on the nitrocellulose support. Anti-TNP serum is then added as a first antibody and incubated so that the first antibody will attach to the TNP-modified proteins. Anti-IgG-peroxidase conjugate is added as a second antibody. Color is then developed to detect the protein. The proteins immobilized on nitrocellulose are directly measured by measuring the resultant color. However, this method suffers from the requirement for antibodies.
U.S. Pat. No. 4,279,885 to Reese et al., describes a solid phase competitive protein binding assay where an antigen or hapten can be assayed. The method involves competition between the analyte and a labeled form thereof for a limited number of receptor or binding sites which are immobilized to a solid support. The assay may be conducted by mixing the components simultaneously or sequentially. The sequential assay involves contacting a solution of an analyte with a support containing immobilized receptors or antibodies, followed by contacting the mixture with a tracer. The tracer may be the analyte, or analog thereof, which contains a label or tag.
Quantitative assays which do not utilize antibodies are preferable. For example, a sample containing protein to be assayed is mixed with a marker protein on contact with a polystyrene latex. A competition is created between the marker enzyme and the analyte protein for the limited surface binding sites. The amount of enzyme remaining in the supernatant is then determined. The inactivation of the enzyme upon binding to the hydrophobic latex surface allows measurement of the bound/free enzyme ratio, and thus, the competing protein concentration. Sandwick, et al., Anal. Biochem. 147:210-216 (1985). However, different proteins have markedly different affinities for solid supports. Thus, it is necessary to construct a standard curve for each protein which is to be quantified. In addition, relatively significant amounts of proteins are required for this assay. Thus, this method is not practical for quantitation of trace quantities of isolated proteins which may comprise only a few micrograms obtained from hundreds of litres of fermentation broth.
Thus, it would be desirable to have a quantitative assay which is fast and reliable, requires only minimal amounts of analyte, and does not chemically modify the analyte.